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Tumor Biology |
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [L. M., C. T., S. H., C. B., M. M., M. B-E.], and Abgenix Inc., Fremont, CA 94555 [L. G., J. M. G., X. F.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4ß1) and the vitronectin receptor (vß3) correlates with progression of human melanoma (3
, 4)
. Other molecules, the expression of which increases with the advanced stage of disease in melanoma, are particular isoforms of CD44, a cell surface glycoprotein that functions as the major receptor for hyaluronate (4)
, HLA-DR, and intracellular adhesion molecule 1 (5)
. CAMs do not, however, simply glue cells together. CAMs are also involved in signal transduction; i.e., upon adhesion to fibronectin, a signal transduction cascade is activated through the phosphorylation of focal adhesion kinase, which in turn activates the RAS pathway of signal transduction (6)
. MCAM, previously known as MUC18, Mel-CAM, or CD146, is a newly recognized CAM belonging to the immunoglobulin superfamily (7, 8, 9) . cDNA cloning and sequencing revealed that MCAM has significant homology to several CAMs of the immunoglobulin superfamily, including BEN (10) , N-CAM (11) , MAG (12) , and DCC (13) . Some of these proteins are known to exhibit changes in their expression pattern in several human tumors. MCAM is an integral membrane glycoprotein with an apparent molecular weight of 113,000. It contains five immunoglobulin-like domains, and its cytoplasmic domain contains several protein kinase recognition motifs, suggesting the involvement of MCAM in cell signaling (9) . MCAM can mediate heterotypic adhesion between cells, although the counter receptor or ligand for MCAM has yet to be identified (14) . The malignant potential of cutaneous melanoma is directly related to the vertical thickness of the lesion (15 , 16) . Analysis of primary melanomas indicates that although the majority of advanced and metastatic tumors strongly express the MCAM antigen, its expression on thin tumors (<0.75 mm), which have only a low probability of metastasizing, and on benign nevi is weaker and less frequent (7 , 17) . In addition, we have previously demonstrated a positive correlation between MCAM expression and the ability of human melanoma cell lines to metastasize in nude mice (18) . Our studies in murine models suggest that MCAM/MUC18 contributes to tumor growth and metastases formation in vivo (19) . Indeed, ectopic expression of MCAM/MUC18 in radial growth phase melanoma cell lines (MCAM negative) resulted in an increase in their tumorigenicity and metastatic potential in nude mice (19) . The transfected cells displayed increased homotypic adhesion, increased attachment to human endothelial cells, up-regulation of the metalloproteinase MMP-2, and increased invasiveness through Matrigel-coated filters (19) . Moreover, it has recently been shown that the production of tumorigenic variants from a nontumorigenic melanoma cell line is accompanied by MCAM up-regulation (20) .
These observations have established MCAM/MUC18 as a candidate mediator of tumor growth, angiogenesis, and metastasis in human melanoma and lend credence to the rationale that blockade of MCAM/MUC18 might be a potential target for immunotherapy against human melanoma.
In the present study we used a fully human anti-MCAM/MUC18 Ab (ABX-MA1; produced by Abgenix) to block the MCAM/MUC18 adhesion molecule on melanoma cells and analyzed its effect on tumor growth, angiogenesis, and metastasis of human melanoma. We show that ABX-MA1 treatment of nude mice bearing melanoma cells suppressed their tumor growth and the formation of metastases. ABX-MA1 disrupted spheroid formation by melanoma cells expressing MCAM/MUC18 (homotypic interaction) and the ability of these cells to adhere to vascular endothelial cells (extravasation). Inhibition of tumor growth and metastasis in vivo correlated with decreased vascularization, attributed in part to a decrease in MMP-2 expression and activity. Moreover, in an in vitro vessel formation assay, ABX-MA1 directly interfered with tube formation by HUVECs. These results suggest that blocking MCAM/MUC18 with ABX-MA1 could be beneficial in treating melanoma patients either alone or in combination with other chemotherapeutic or antiangiogenic agents.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Male athymic BALB/c nude mice were purchased from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used at 8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and NIH.
Fully Human Anti-MUC18 Ab (ABX-MA1).
ABX-MA1 is a human IgG2 monoclonal Ab directed against human MUC18/MCAM that was generated using Abgenix’s proprietary XenoMouse mice. In XenoMouse technology, murine heavy and light chain loci have been inactivated and subsequently replaced with a majority of human heavy and 
light chain immunoglobulin loci. When immunized, these mice produce fully human Abs (26)
. The mice used for this immunization contained only the human IgG2 heavy chain sequences and human light chain. ABX-MA1 detects a single specific Mr 113,000 MCAM/MUC18 band in Western blot analysis and binds specifically to MCAM/MUC18 as determined by FACS analysis with metastatic melanoma cells. Chemopure human IgG control Ab was purchased from Jackson ImmunoResearch (West Grove, PA) and used at the same concentration as ABX-MA1 in all experiments.
Western Blot Analysis.
Melanoma cell lines were seeded at 1 x 106 in 100-mm tissue culture plates in 10 ml of CMEM. After overnight incubation, the plates were washed two times in PBS and scraped off in 400 µl of Triton lysis buffer, 1 µl of DTT, and 4 µl of protease inhibitor mixture. After a 30-min incubation on ice, the cells were centrifuged at 15,000 rpm for 15 min. The protein concentration was determined (Bio-Rad and BSA standards), and 40 µg of protein were loaded onto a 10% SDS-PAGE gel and electrophoretically transferred to a 0.45-µm nitrocellulose membrane (Millipore, Bedford, MA). The membrane was blocked with 5% milk in tween tris buffered saline for 1 h. Primary incubation of both cell lines was accomplished by cutting the membranes and incubating them in 1 ml of either control IgG (1:500 dilution) or ABX-MA1 overnight. Membranes were probed with secondary Ab peroxidase-conjugated AffiniPure rabbit antihuman IgG (H+L) for 1 h and then washed with tween tris buffered saline. Probed proteins were detected by enhanced chemiluminescence (Amersham) following the manufacturer’s protocol.
Flow Cytofluorometry.
Quantitative analysis of MUC18 on cell surfaces was determined by FACS analysis. A375SM and SB-2 cells (1 x 105) were plated in 6-well plates (Fisher Scientific, Houston, TX) and incubated for 24 h. After attachment, wells were scraped with a disposable cell scraper (Sarstedt) and incubated with control IgG or ABX-MA1 for 1 h at 4°C. After several washings with FACS buffer, all samples were incubated with R-phycoerythrin-conjugated AffiniPure F(ab')2 fragment goat antihuman IgG (H+L) [1:200 dilution (Jackson ImmunoResearch)] for 1 h at 4°C in the dark. The cells were fixed in 1% paraformaldehyde in PBS and examined by cytofluorometry.
Three-dimensional Spheroid Culture.
Multicellular spheroids were generated by the liquid overlay technique (27
, 28)
. Briefly, 24-well tissue culture plates (Costar) were coated with 250 µl of prewarmed 1% SeaPlaque agarose (FMC Bioproducts, Rockland, ME) solution in serum-free MEM. After the agarose was allowed to solidify and form a thin layer on the bottom of the dish, a single-cell suspension of A375SM or SB-2 (1 x 105) was diluted in 25 µl of hybridoma medium, plated with 475 µl of control IgG (1:200 dilution) or ABX-MA1, and incubated at 37°C in 5% CO2/95% air. After 24 h, spheroid formation was determined. Images were captured by bright-field microscopy and photographed in digital format.
Attachment of Melanoma Cells to HUVECs.
Attachment of melanoma cells to endothelial cells treated with 12 µg/ml control IgG or ABX-MA1 was measured by plating 4 x 104 HUVECs in 96-well plates and allowing them to attach for 24 h. A thin overlay of 2% BSA was placed in each well and incubated overnight at 37°C. Then 5 x 104 cells with or without treatment were added to each well and incubated overnight at 37°C. Wells were rinsed twice with PBS, and cells in each well were counted. Results are presented as the percentage of cells adhered from the total number of cells seeded.
Effect of ABX-MA1 on Proliferation.
Cells (2000 melanoma cells/well in 96-well plates) were treated with 100 µg/ml ABX-MA1, IgG control Ab, or CMEM for 5 days and then analyzed for viability by MTT assay (which determines relative cell numbers based on the conversion of MTT to formazan in viable cells). MTT (40 µg/ml) was added to each well and incubated for 2 h. The medium was removed, and 100 µl of DMSO were added to lyse cells and solubilize formazan. Absorbance was determined on a microplate reader.
In Vivo Tumor Growth and Metastasis.
To prepare tumor cells for inoculation, cells in exponential growth phase were harvested by brief exposure to a 0.25% trypsin/0.02% EDTA solution (w/v). The flask was tapped sharply to dislodge the cells, and supplemented medium was added. The cell suspension was pipetted to produce a single-cell suspension. The cells were washed and resuspended in Ca2+/Mg2+-free HBSS to the desired cell concentration. Cell viability was determined by trypan blue exclusion, and only single-cell suspensions of >90% viability were used. s.c. tumors were produced by injection of 1–5 x 105 tumor cells in 0.2 ml of HBSS over the right scapular region. Growth of s.c. tumors was monitored by weekly examination of the mice and measurement of tumors with calipers. The mice were killed 5 weeks after injection, and tumors were processed for H&E staining.
For experimental lung metastasis, 1 x 106 tumor cells/0.2 ml HBSS were injected into the lateral tail vein of nude mice. The mice were killed after 60 days, and the lungs were removed, washed in water, and fixed with Bouin’s solution for 24 h to facilitate counting of tumor nodules as described previously (29) . The number of surface tumor nodules was counted under a dissecting microscope. Sections of the lungs were stained with H&E to confirm that the nodules were melanoma and to monitor the presence of micrometastases. Both s.c. and i.v. groups were treated once or twice weekly with either 1 mg or 100 µg of ABX-MA1 as indicated or control IgG Ab by i.p. injection.
Zymography.
MMP-2 activity was determined on substrate-impregnated gels (30)
with minor modifications. We plated 5 x 103 metastatic A375SM cells in 6-well plates and allowed them to attach for 24 h. Cells were treated with 100 µg/ml ABX-MA1, control IgG, or CMEM for 5 days. Treatment for 5 days was found to be optimal for the Ab to affect MMP-2 activity. On day 5, CMEM was removed and replaced with serum-free medium overnight. The supernatant was collected, the volume was adjusted for cell number, and the supernatant was loaded on gelatin-impregnated (1 mg/ml; Difco, Detroit, MI) SDS-8% polyacrylamide gels under nonreducing conditions, followed by 30 min of shaking in 2.5% Triton X-100 (BDH, Poole, United Kingdom). The gels were then incubated for 16 h at 37°C in 50 mM Tris, 0.2 M NaC1, 5 mM CaC12, and 0.02% Brij 35 (w/v) at pH 7.6. At the end of the incubation, the gels were stained with 0.5% Coomassie G 250 (Bio-Rad, Richmond, CA) in methanol/acetic acid/H2O (30:10:60). The intensity of the various bands was determined on a computerized densitometer (Molecular Dynamics type 300A).
Invasion Assay through Matrigel.
Invasion of highly metastatic A375SM and WM2664 cells was measured by plating 2.5 x 103 cells on 6-well plates and allowing them attach for 24 h. After 5 days of treatment with 100 µg/ml ABX-MA1, control IgG, or CMEM, cells were released from the plates by a brief exposure to trypsin-EDTA (Life Technologies, Inc.), counted, and centrifuged. Biocoat Matrigel invasion chambers (Becton-Dickinson) were primed according to the manufacturer’s directions. A solution of 20% CMEM was placed in the lower well to act as a chemoattractant, and 2.5 x 103 cells in 500 µl of serum-free medium with appropriate Ab were placed in the upper chamber of the Matrigel plate and incubated at 37°C for 22 h. Cells on the lower surface of the filter were stained with Diff-Quick (American Scientific Products, McGraw Park, IL) and quantified with an image analyzer (Optimas 6.2) attached to an Olympus CK2 microscope. The data were expressed as the average number (±SD) of cells from 10 fields that migrated to the lower surface of the filter from each of three experiments performed.
Transient Transfection and Luciferase Assays: Effect of ABX-MA1 on MMP-2 Promoter.
The MMP-2 promoter construct was generated by cutting the MMP-2 promoter region, -390 to +290 (31)
, out of p682 basic (chloramphenicol acetyltransferase-driven MMP-2 promoter; Ref. 30
) at the HindII/XbaI sites and ligating it into pGEM-9Zf(-) vector (Promega, Madison, WI) using the same sites. The MMP-2 promoter region was then removed via the SpeI/SalI sites and ligated into the pGL3-Enhancer (Promega; Ref. 32
). Melanoma cells were treated with 100 µg/ml ABX-MA1, control IgG, or CMEM for 4 days and then transfected with 10 ng of pB-actin-RL (33)
and 2 µg of plasmid DNA of either luciferase basic vector, SV40 positive control, or MMP-2 promoter vector, using 10 µl of Lipofectin reagent (Life Technologies, Inc.). After 12 h, the medium was changed, and treatments were added. Cells were lysed and analyzed using dual luciferase assay (Promega) and Ascent Lumiskan plate reader and software (33)
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Effect of ABX-MA1 on Vessel-like Tube Formation by HUVECs.
The basement membrane-like substrate (Matrigel) induces HUVECs to rapidly form vessel-like tubes in vitro (34)
. Because MUC18/MCAM is also expressed by HUVECs, we analyzed the effect of ABX-MA1 on HUVEC tube formation. To that end, 24-well plates were coated with reconstituted Matrigel (Beckett-Dickenson) following the manufacturer’s directions. HUVECs were pretreated with medium containing 100 µg/ml ABX-MA1, 100 µg/ml IgG, or MCDB (FIND) alone for 4 days. Treatment for 4 days was found to be optimal for the Ab to affect tube formation. Cells were briefly trypsinized, and 2 x 104 cells were added to each well of and incubated at 37°C, in 5% CO2, for 8 h. Pictures were captured with bright-field microscopy using a Sony digital camera equipped with the Opitmas 6.2 program.
In Situ TUNEL Assay.
Tissues were fixed in 10% buffered formalin solution and then embedded in paraffin. Thin sections (4 µm) were prepared, and the TUNEL assay was performed using a commercial kit according to the manufacturer’s protocol (Promega). Briefly, tissue sections were deparaffinized and fixed at room temperature for 5 min in 4% paraformaldehyde. Cells were stripped of proteins by incubation for 10 min with 20 µg/ml proteinase. The tissue sections were then permeabilized by incubating them with 0.5% Triton X-100 in PBS for 5 min at room temperature. After being rinsed twice with PBS for 5 min, the slides were incubated with terminal deoxynucleotidyl transferase buffer for 10 min. Terminal deoxynucleotidyl transferase and buffer were then added to the tissue sections, which were incubated in a humid atmosphere at 37°C for 1 h. The slides were washed three times with PBS for 5 min. Prolong solution (Molecular Probes, Eugene, OR) was used to mount the coverslips. Immunofluorescence microscopy was performed using a x40 objective (Zeiss Plan-Neofluar) on an epifluorescence microscope equipped with narrow bandpass excitation filters mounted on a filter wheel (Lud1 Electronic Products, Hawthorne, NY) to select for green fluorescence. Images were captured using a cooled charge-coupled device camera (Photometrics, Tucson, AZ) and SmartCapture software (Digital Scientific, Cambridge, United Kingdom) on a Macintosh computer. Images were further processed using Adobe PhotoShop software (Adobe Systems, Mountain View, CA). Quantitation of TUNEL was determined by conventional 3,3'-diaminobenzidine staining. Results are presented as the mean percentage ± SD of apoptotic cells from the total number of cells counted in 8 fields/slide.
IHC.
For CD31 and MMP-2 staining, sections of frozen tissues were prepared from tumor xenografts. The slides were then rinsed twice with PBS, and endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 min. The samples were then washed three times with PBS and incubated for 10 min at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 h at 4°C with a 1:100 dilution of monoclonal rat anti-CD31 (1:100) Ab or anti-MMP-2 (1:100; PharMingen, San Diego, CA). The samples were then rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution of peroxidase-conjugated antimouse IgG1, antirabbit IgG, or antirat IgG. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics, Huntsville, AL). The sections were then washed three times with distilled water and counterstained with Gill’s hematoxylin. Sections (4-µm thick) of formalin-fixed, paraffin-embedded tumors were also stained with H&E for routine histological examination.
Statistical Analysis.
The in vitro data were analyzed for significance by using Student’s t test (two-tailed), and the in vivo data were analyzed by using the Mann-Whitney test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clone 3.19.1 was chosen for further in vitro and in vivo analyses and designated ABX-MA1. ABX-MA1 recognized and detected a single band corresponding to the Mr 113,000 MUC18 protein in the metastatic melanoma cell lines A375SM and WM2664 [both expressed high levels of MUC18 (Ref. 19
; Fig. 1A
, Lanes 1 and 4)]. This Ab failed to detect any MUC18 protein in the nonmetastatic melanoma cell line SB-2 [MUC 18 negative (Ref. 19
; Fig. 1A
, Lane 2)]. The specificity of the Ab was further confirmed by detection of the Mr 113,000 MUC18 band in SB-2 cells after transfection with the MUC18 gene as we have described previously (Ref. 19
; Fig. 1A
, Lane 3). In addition to metastatic melanoma cells, MUC18 is also expressed on HUVECs (7
, 8)
. ABX-MA1 detected the Mr 113,000 protein on HUVECs (Fig. 1A
, Lane 6), but not on murine nude mouse endothelial cells (NME, Lane 5). The MUC18 adhesion molecule was also detected with ABX-MA1 by FACS analysis on the cell surface of A375SM cells, but not on SB-2 cells (Fig. 1B)
. Thus, ABX-MA1 specifically bound and detected the Mr 113,000 MUC18 receptor on metastatic human melanoma and HUVECs.
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. When we cultured the cells with ABX-MA1 (added at time 0), spheroid formation was disrupted only in A375SM cells and not in SB-2 cells. This effect was not observed when control IgG Ab was added to the cultures (Fig. 2)
. This assay confirms the ability of ABX-MA1 to disrupt spheroid formation by metastatic MUC18-positive melanoma cells.
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. In contrast, treatment with ABX-MA1 reduced tumor growth to approximately 400 mm3 for WM2664 and 180 mm3 for A375SM during the same periods of time (Fig. 3)
.
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, both the incidence and number of lung metastases of both melanoma cell lines were lower in ABX-MA1-treated mice than in the control IgG-treated group. Both doses of ABX-MA1 were equally effective in inhibiting the formation of metastases. In IgG-treated mice, A375SM cells produced numerous lung metastases (median, 17; range, 5–34), whereas the WM2664 cells produced a median of 11 lung colonies (range, 4–21). In contrast, treatment with ABX-MA1 significantly inhibited the ability of both A375SM and WM2664 cells to form metastases in nude mice (median, 1–2; range, 0–8; P < 0.001). Collectively, these data demonstrate that treatment of mice with ABX-MA1 suppressed melanoma tumor growth and metastasis.
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. These data suggest that ABX-MA1 did not affect melanoma cell proliferation in vitro. ABX-MA1 also did not alter the proliferation rate of HUVECs in vitro (Fig. 4C)
.
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, the Mr 72,000 MMP-2 collagenase activity of ABX-MA1-treated A375SM cells was significantly lower than that of either IgG-treated or untreated (grown in CMEM) control cells. Because MMP-2 is the major metalloproteinase expressed and secreted by melanoma cells, we observed a faint band of MMP-9 activity in all melanoma cells tested, regardless of treatment with ABX-MA1 (data not shown).
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. These results suggest that MUC18 may directly regulate MMP-2 expression at the transcriptional level and that blocking of MUC18 by ABX-MA1 suppressed MMP-2 expression and activity in melanoma cells.
Suppression of Human Melanoma Cell Invasion by ABX-MA1.
We next analyzed whether the decreased expression of MMP-2 in ABX-MA1-treated cells correlated with their ability to invade through the basement membrane, an important component in the process of tumor invasion and metastasis. To that end, 2.5 x 103 A375SM or WM2664 melanoma cells that had been treated with 100 µg/ml ABX-MA1 or control IgG Ab for 5 days were placed in the upper compartment of an invasion chamber in the presence of 100 µg/ml ABX-MA1 or control IgG. After 22 h of incubation, the cells on the lower surface of the filters were counted. A375SM and WM2664 cells treated with ABX-MA1 exhibited significantly less invasion through Matrigel-coated filters than IgG-treated or untreated cells [2068 ± 129 versus 57 ± 8 (number of migrated cells ± SD) for A375SM (P < 0.001) and 1866 ± 130 versus 56 ± 7 (P < 0.001) for WM2664; Table 2
]. These results indicate that blockade of MUC18 in melanoma cells by ABX-MA1 inhibited the ability of cells to penetrate through the basement membrane. Collectively, our data indicate that inactivation of MMP-2 by ABX-MA1 in melanoma cell may account for the decrease in their metastatic potential.
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, MMP-2 staining was strong in IgG-treated A375SM and WM2664 tumors but was considerably decreased in ABX-MA1-treated tumors. Thus, blocking of MUC18 by ABX-MA1 Ab in melanoma cells inhibited expression of the MMP-2 gene in vivo as well as in vitro.
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, we found a significant reduction in tumor MVD/field after treatment with ABX-MA1 as compared with control IgG-treated tumors. The mean MVD was 8.1 ± 5.1 in ABX-MA1-treated A375SM tumors and 20.1 ± 3.3 in ABX-MA1-treated WM2664 tumors. In contrast, the mean MVD was 22.2 ± 3.1 and 36.1 ± 5.8 for control IgG-treated A375SM and WM2664 tumors, respectively. Moreover, the number of TUNEL-positive tumor cells was inversely correlated with MVD in the studied tumors. The number of tumor cells undergoing apoptosis was higher in the ABX-MA1-treated animals than in tumors derived from control IgG-treated mice (Fig. 7)
. Quantitative analysis of the TUNEL assay has revealed that the percentage of apoptotic cells was 20 ± 4.8% in ABX-MA1-treated A375SM and 18.6 ± 5.3% in ABX-MA1-treated WM2664 tumors. In contrast, the percentage of apoptotic cells was 1 ± 0.2% and 1.2 ± 0.3% for control IgG-treated A375SM and WM2664 tumors, respectively. These data indicate that ABX-MA1 treatment significantly decreased tumor-associated neovascularization and subsequently increased apoptosis of tumor cells.
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) and because melanoma cells interact with vascular endothelial cells through MUC18-MUC18 interactions, we next examined the ability of ABX-MA1 to block the attachment of melanoma cells to HUVECs. To that end, the attachment of SB-2 (MUC18-negative) and A375SM (MUC18-positive) cells to HUVECs was analyzed in the presence of ABX-MA1 or IgG control Ab. Fig. 8
shows that very few SB-2 melanoma cells attached to HUVECs and that the addition of control IgG or ABX-MA1 Ab to the cultures did not alter SB-2-HUVEC interactions (Fig. 8A)
. In contrast, A375SM cells attached to HUVECs, and treatment with ABX-MA1, but not with control IgG Ab, inhibited A375SM-HUVEC interaction (Fig. 8A)
. A quantitative summary of three experiments performed on the attachment of A375SM to HUVECs (Fig. 8B)
demonstrates that ABX-MA1 reduced the interaction of A375SM with HUVECs by 80%. These data provide a further potential mechanism by which ABX-MA1 may inhibit extravasation and hence metastasis.
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, CMEM-pretreated HUVECs formed lumen-like structures and anastomosing tubes with multicentric junctions (Fig. 9A)
. Similar endothelial cell structural morphogenesis occurred in IgG-pretreated HUVECs (Fig. 9B)
. In contrast, vessel-like tube formation was dramatically disrupted in HUVECs pretreated with ABX-MA1 (Fig. 9C)
. When ABX-MA1 was added to the cultures after tube formation by HUVECs had occurred, the morphogenesis of preexisting vessel-like tubes was not altered (Fig. 9D)
. Thus, ABX-MA1 directly inhibited de novo formation of capillary-like networks but did not inhibit preexisting tubule networks.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whereas ABX-MA1 did not alter the proliferation rate of melanoma cells in vitro, it had a profound effect on their tumorigenicity and metastasis potential in vivo. These data indicate that the role of MUC18/MCAM in the progression of human melanoma is more complex than simple autocrine growth stimulation or homotypic interactions.
Indeed, we showed that MUC18/MCAM had a significant effect on angiogenesis and invasion, although the ligand for MUC18/MCAM has yet to be identified. Growth and metastasis of human melanoma cells depend on their ability to develop an adequate vasculature. In fact, the progression of neoplasms from the benign to malignant state is often associated with a switch to an "angiogenic phenotype" representing an increase in proangiogenic molecules produced by the tumor cells and organ-specific environments (36 , 37) . Melanoma cells secrete a variety of proangiogenic molecules, including basic fibroblast growth factor (38 , 39) , vascular endothelial growth factor (40) , and interleukin 8 (24 , 30) . MUC18/MCAM can indirectly affect angiogenesis and invasion of human melanoma cells through up-regulation of MMP-2 and cell interaction with the extracellular matrix and vascular endothelial cells. We found that blockade of MUC18/MCAM by ABX-MA1 suppressed angiogenesis of human melanoma cells grown in nude mice. All of the control tumors were highly vascularized and produced large tumors, whereas the ABX-MA1-treated mice produced small tumors. The retarded tumor growth was directly correlated with decreased blood vessel formation and an increased number of apoptotic tumor cells (TUNEL positive). The inhibition of angiogenesis could be attributed to down-regulation of MMP-2. The proteolytic effect of MMPs facilitates the migration of endothelial cells through the altered extracellular matrix toward the source of the angiogenic stimulus; in this manner, MMP-2 is an integral component of the angiogenesis pathway (41) . However, we cannot rule out the possibility that ABX-MA1 can activate murine complement or mediate antibody dependent cell cytotoxicity with murine monocytes through the Fc portion of the Ab. These possibilities are currently under investigation in our laboratory.
In addition to the tumor cells themselves, ABX-MA1 may also affect the host vascular endothelial cells, integrity, formation of new blood vessels, and their interaction with melanoma cells. Because HUVECs also express MUC18/MCAM, ABX-MA1 was found to inhibit their interaction with MUC18-positive metastatic melanoma cells. The inhibition of this interaction could affect extravasation, an important step in the process of metastasis.
Moreover, in an in vitro endothelial cell morphogenesis assay (vessel-like tube formation assay), we found that ABX-MA1 directly inhibited the formation of a capillary-like network by HUVECs. Treatment with ABX-MA1 did not affect existing vessel-like tubes in vitro, disrupting only the formation of the newly formed blood vessels. This observation is of great importance for clinical implications.
The potential of Ab therapy represented by ABX-MA1 fits recent discoveries. Ab immunotherapy provides a novel approach for the treatment of a broad spectrum of diseases including cancer (42, 43, 44, 45, 46) . Cetuximab (IMC-C225), a mouse-human chimeric anti-EGF receptor monoclonal Ab, and ABX-EGF have been shown to inhibit the proliferation of a variety of cultured human tumor cell lines that overexpress EGF receptor and to inhibit tumor growth in several xenograft models (45 , 46) .
Currently, both of these Abs are being evaluated in clinical trials. The therapeutic modalities to control tumor growth and metastasis of human melanoma are very limited. The idea of using fully humanized Abs to block MUC18 is especially appealing because multiple dose regimens of the Ab could be administered to the patients with little risk of mounting an immune reaction. Our studies should promote serious consideration for initiating a Phase I/II clinical trail with ABX-MA1 in patients with metastatic melanoma. ABX-MA1 can be used either alone or in combination with chemotherapy or other anticancer agents to increase the efficacy of the treatment. In addition, ABX-MA1 should be considered as a treatment modality for other solid tumors in which MUC18/MCAM may play an angiogenic role, including prostate cancer (47) .
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FOOTNOTES |
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1 Supported in part by NIH Grant CA 76098. 
2 To whom requests for reprints should be addressed, at Department of Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-4004; Fax: (713) 792-8747; E-mail: mbareli{at}mail.mdanderson.org
3 The abbreviations used are: CAM, cell adhesion molecule; FACS, fluorescence-activated cell-sorting; HUVEC, human umbilical vein endothelial cell; IHC, immunohistochemistry; MMP, matrix metalloproteinase; CMEM, complete minimal essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; Ab, antibody; MVD, microvessel density; EGF, epidermal growth factor.
Received 3/27/02. Accepted 7/ 2/02.
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S. Bidlingmaier, J. He, Y. Wang, F. An, J. Feng, D. Barbone, D. Gao, B. Franc, V. C. Broaddus, and B. Liu Identification of MCAM/CD146 as the Target Antigen of a Human Monoclonal Antibody that Recognizes Both Epithelioid and Sarcomatoid Types of Mesothelioma Cancer Res., February 15, 2009; 69(4): 1570 - 1577. [Abstract] [Full Text] [PDF]
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F. I. Staquicini, A. Tandle, S. K. Libutti, J. Sun, M. Zigler, M. Bar-Eli, F. Aliperti, E. C. Perez, J. E. Gershenwald, M. Mariano, et al. A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans Cancer Res., October 15, 2008; 68(20): 8419 - 8428. [Abstract] [Full Text] [PDF]
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S. Vicent, D. Luis-Ravelo, I. Anton, I. Garcia-Tunon, F. Borras-Cuesta, J. Dotor, J. De Las Rivas, and F. Lecanda A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism Cancer Res., April 1, 2008; 68(7): 2275 - 2285. [Abstract] [Full Text] [PDF]
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D. Becker, M. C. Mihm, S. M. Hewitt, V. K. Sondak, J. W. Fountain, and M. Thurin Markers and Tissue Resources for Melanoma: Meeting Report. Cancer Res., November 15, 2006; 66(22): 10652 - 10657. [Abstract] [Full Text] [PDF]
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 P. Bu, L. Gao, J. Zhuang, J. Feng, D. Yang, and X. Yan Anti-CD146 monoclonal antibody AA98 inhibits angiogenesis via suppression of nuclear factor-{kappa}B activation. Mol. Cancer Ther., November 1, 2006; 5(11): 2872 - 2878. [Abstract] [Full Text] [PDF]
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 L. Chin, L. A. Garraway, and D. E. Fisher Malignant melanoma: genetics and therapeutics in the genomic era. Genes & Dev., August 15, 2006; 20(16): 2149 - 2182. [Abstract] [Full Text] [PDF]
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 M. Cerletti, M. J. Molloy, K. K. Tomczak, S. Yoon, M. F. Ramoni, A. T. Kho, A. H. Beggs, and E. Gussoni Melanoma cell adhesion molecule is a novel marker for human fetal myogenic cells and affects myoblast fusion J. Cell Sci., August 1, 2006; 119(15): 3117 - 3127. [Abstract] [Full Text] [PDF]
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V. O. Melnikova, A. A. Mourad-Zeidan, D. C. Lev, and M. Bar-Eli Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis J. Biol. Chem., February 3, 2006; 281(5): 2911 - 2922. [Abstract] [Full Text] [PDF]
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 A. Ebrahimnejad, T. Streichert, P. Nollau, A. K. Horst, C. Wagener, A.-M. Bamberger, and J. Brummer CEACAM1 Enhances Invasion and Migration of Melanocytic and Melanoma Cells Am. J. Pathol., November 1, 2004; 165(5): 1781 - 1787. [Abstract] [Full Text] [PDF]
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A. Rump, Y. Morikawa, M. Tanaka, S. Minami, N. Umesaki, M. Takeuchi, and A. Miyajima Binding of Ovarian Cancer Antigen CA125/MUC16 to Mesothelin Mediates Cell Adhesion J. Biol. Chem., March 5, 2004; 279(10): 9190 - 9198. [Abstract] [Full Text] [PDF]
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N. Rousselet, L. Mills, D. Jean, C. Tellez, M. Bar-Eli, and R. Frade Inhibition of Tumorigenicity and Metastasis of Human Melanoma Cells by Anti-Cathepsin L Single Chain Variable Fragment Cancer Res., January 1, 2004; 64(1): 146 - 151. [Abstract] [Full Text] [PDF]
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 E. C. McGary, A. Heimberger, L. Mills, K. Weber, G. W. Thomas, M. Shtivelband, D. C. Lev, and M. Bar-Eli A Fully Human Antimelanoma Cellular Adhesion Molecule/MUC18 Antibody Inhibits Spontaneous Pulmonary Metastasis of Osteosarcoma Cells In Vivo Clin. Cancer Res., December 15, 2003; 9(17): 6560 - 6566. [Abstract] [Full Text] [PDF]
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T. Ishida, R. K. Kundu, E. Yang, K.-i. Hirata, Y.-D. Ho, and T. Quertermous Targeted Disruption of Endothelial Cell-selective Adhesion Molecule Inhibits Angiogenic Processes in Vitro and in Vivo J. Biol. Chem., September 5, 2003; 278(36): 34598 - 34604. [Abstract] [Full Text] [PDF]
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 X. Yan, Y. Lin, D. Yang, Y. Shen, M. Yuan, Z. Zhang, P. Li, H. Xia, L. Li, D. Luo, et al. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth Blood, July 1, 2003; 102(1): 184 - 191. [Abstract] [Full Text] [PDF]
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 L. Grumolato, A. G. Elkahloun, H. Ghzili, D. Alexandre, C. Coulouarn, L. Yon, J.-P. Salier, L. E. Eiden, A. Fournier, H. Vaudry, et al. Microarray and Suppression Subtractive Hybridization Analyses of Gene Expression in Pheochromocytoma Cells Reveal Pleiotropic Effects of Pituitary Adenylate Cyclase-Activating Polypeptide on Cell Proliferation, Survival, and Adhesion Endocrinology, June 1, 2003; 144(6): 2368 - 2379. [Abstract] [Full Text] [PDF]
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